Organic dyes, such as fluorescent molecules, have been used to label biological materials. These fluorochromes, however, have several disadvantages. For example, fluorochromes generally have narrow wavelength bands of absorption (e.g., about 30-50 nm), broad wavelength bands of emission (e.g., about 100 nm), and broad tails of emission (e.g., another 100 nm) on the red side of the spectrum. Due to the wavelength properties of these fluorophores, the ability to use a plurality of different colored fluorescent molecules is severely impaired. Furthermore, the fluorescence is extremely susceptible to photobleaching.
Nanometer-size semiconductor particles (nanoparticles) are particles which demonstrate quantum confinement effects in their luminescent properties. These semiconductor nanoparticles are also known as “quantum dots.” Colloidal particles containing quantum dots can be excited by a single excitation source to provide extremely robust, broadly tunable nanoemitters. In addition, the nanoparticles exhibit optical properties which are superior to that of organic dyes. Therefore, due to their distinctive luminescent properties, quantum dots have the potential to dramatically improve the use of fluorescent markers in biological studies.
However, before nanoparticles can be widely used as biological labels, they must maintain several key properties under aqueous biological conditions: solubility, low toxicity, efficient fluorescence, colloidal stability, and low non-specific adsorption. Unfortunately, despite recent advances, these conditions have not been simultaneously satisfied by the prior art. Not surprisingly, no reports have demonstrated in vivo applications of semiconductor nanoparticles.
It has proven especially difficult to render semiconductor nanoparticles soluble in water. Currently, the main strategy to solubilize (e.g., dissolve) semiconductor nanoparticles in water is to exchange the hydrophobic ligands (e.g., organic moieties which are insoluble in water) present after synthesis of the semiconductor nanoparticles with thiolated hydrophilic ligands. Two approaches have been used.
In the first approach, the hydrophobic ligands surrounding the nanoparticle are exchanged by a monolayer of ligands which is constituted of a thiol group at one end that binds to the nanoparticle surface and a hydrophilic group at the other end (Chan et al. Science (1998) 281:2016; Mikulec, F. PhD. thesis at the Massachusetts Institute of Technology, 1999; Bawendi et al. U.S. Pat. No. 6,319,426 B1). However, this process yielded semiconductor nanoparticles with poor stability.
In the other approach, the hydrophobic ligands are exchanged by a multilayered shell of crosslinked silanes (Bruchez et al. Science (1998) 281:2013). One disadvantage to this approach is the long amount of time it takes to exchange the hydrophobic ligands with crosslinked silanes. In addition, the soluble semiconductor nanoparticles exhibit high non-specific adsorption (e.g., the soluble semiconductor nanoparticles aggregate in biological systems).
The use of micelles to solubilize semiconductor nanoparticles in water is disclosed in U.S. Pat. No. 6,319,426 B1 to Bawendi et al. A micelle is typically a colloidal aggregate of amphiphilic substances. Generally, in aqueous medium, the nonpolar (e.g., hydrophobic) ends of the amphiphilic substance face inward and the polar (e.g., hydrophilic) tails face outward. This orientation of the amphiphilic substance results in a micelle having a hydrophobic core and a hydrophilic shell. Thus, unfavorable contacts between water and the hydrophobic tails are eliminated.
The micelles disclosed in the Bawendi et al. patent are formed using sodium dioctyl sulfosuccinate (AOT) or Brij surfactants. The AOT reagent contains two short hydrophobic chains (each chain containing eight carbon atoms) and an ion for the hydrophilic portion. The Brij surfactants contain block copolymers that have a long hydrophilic polyethylene glycol chain and one long hydrophobic hydrocarbon chain (containing twelve or eighteen carbon atoms). However, the micelles that are formed using the reagents disclosed in the Bawendi et al. patent are not stable in aqueous solutions.
Therefore, there is a need for stable, water soluble semiconductor nanoparticles, which can be used in biological applications, both in vitro and in vivo. The absorption and emission properties of the stable, water soluble semiconductor nanoparticles offer advantages over current conventional organic dyes.